Substrates for high-density cell growth and metabolite exchange

ABSTRACT

A polymer or other substrate optimized for growing cells is described, which takes the form of a micro-thin bag with gas permeable sides. Sides of the bag can be held at a fixed distance from one another with a multitude of tiny micropillars or other spacers extending between them, keeping the bag at a predetermined thickness and preventing the bag from collapsing and the sides from sticking together. In other embodiments, the sides may be held apart by gas pressure alone. A 0.01 μm to 1000 μm parylene or other biocompatible coating over the bag outsides controls the permeability of the bag material and provides a bio-safe area for cell growth. An alternate configuration uses open-cell foam with skins coated with a biocompatible coating. Tubes going into multiple bags can be connected to a manifold that delivers gaseous oxygen or removes carbon dioxide and other waste gases. Multiple bags can be stacked together tightly, with o-ring spacers in between, and housed within a vessel to form a high-surface area, ultra-compact cell growing system. For cells growing on the bags, liquid nutrients can be fed by way of the tube spacers, and oxygen and waste gases permeated through the bag sides and transported within the bags.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/647,156, filed Jul. 11, 2017 (U.S. Pat. No. 10,053,660, issued Aug.21, 2018), which claims the benefit of U.S. Provisional Application No.62/361,390, filed Jul. 12, 2016. The above applications are herebyincorporated in their entireties for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

NOT APPLICABLE

BACKGROUND 1. Field of the Art

Embodiments of the present invention generally relate to a bioreactorapparatus for culturing microorganisms and growing cells, including gaspermeable non-collapsible and/or non-expandable bags withmicrofabricated features and coatings, as well as methods of manufactureand use.

2. Description of the Related Art

Large-scale high density cell culture is important for manybiotechnology applications where cells are used to produce specificmolecules, proteins, viruses, or other products. Increasing cell densityallows for greater production per unit volume, which can help reducecosts through space savings and more concentrated product.

The challenge of high-density cell growth arises from mass transportlimitations particularly with respect to oxygen, nutrients, and wasteproducts. In low-density cell culture systems, passive diffusion ofmetabolites is often sufficient to meet the metabolic demands of cells;however, in high-density cell culture systems, the metabolic demand ofcells exceeds supply from diffusion alone, requiring additional masstransport mechanisms such as convection.

The additional requirement of high-density cell culture is a highsurface area to volume ratio, particularly for adherent cellpopulations. This is because many cells grow in monolayers, and theirgrowth is inhibited once they reach confluence.

Several technologies have been developed to enhance cell densityincluding cell factory systems, wave/stirred bioreactors withmicrocarriers, and perfused dialysis membrane systems.

Cell factory systems are most similar to conventional flask culturesystems except that cell factory systems contain multiple layers ofgrowth substrate within a single flask. The cell density that can beachieved is not very high due to the large spacing between layers,resulting in a low surface-to-volume ratio, and the reliance ondiffusive transport for all metabolites.

Wave and stirred bioreactor systems add convection to enhance masstransport by gently mixing cell microcarriers, small neutrally buoyantparticles with surface chemistry suitable for cell adhesion and growth,within a container of media. The combination of high surface areaafforded by the microcarriers and the convective mixing allows forhigher cell densities to be achieved compared to cell factory systems.However, convective mixing also causes shear forces on the cells, whichcan induce cell death, thereby limiting the degree of mixing and masstransport to cells that can be achieved. Such systems are oftenshear-limited due to the need to enhance mass transport through mixingrather than surface area limited.

Perfused dialysis membrane systems overcome the shear problem byperfusing gas through tightly packed semi-permeable dialysis tubes andallowing diffusion to deliver oxygen to cells. However, the geometry ofthe dialysis tubes prevents very high surface areas to volume ratiosfrom being achieved. Furthermore, there are challenges with cell removalfrom the highly porous membranes on which the cells grow.

In summary, some challenges of high density cell growth include: 1)achieving high growth surface area to volume ratio; 2) maintainingadequate metabolite transport within the system; and 3) maintainingshear forces experienced by cells below lethal levels. While severalexisting technologies have attempted to overcome these challenges, thereremains extensive room for improvement of the art.

BRIEF SUMMARY

Generally, very thin gas permeable plastic bags with an array ofinternal connection points, such as microfabricated weld points toprevent expansion and/or internal spacers to prevent collapse, arepresented. The bags are coated with a thin (e.g., 2 μm and 10 μm thick)biocompatible coating, such as parylene, to control permeability andprovide a surface for cells to adhere. The biocompatible coating may besubject to an oxygen plasma and/or ammonia plasma treatment, and furthercoatings of agarose, etc. may be applied in order to further improvecell adhesion.

Open cell foam with a skin may also be formed into an expansionresistant, collapse resistant bag. The outside skin is layered with abiocompatible coating to control permeability and provide a cleansurface for cell growth, and the edged sealed to form a bag. An inletallows air, oxygen, or other gases into the bag's interior.

In use, the bag is immersed in a nutrient-rich liquid water culture andprovided with or otherwise inflated with gaseous oxygen to act as asubstrate for cell growth. A cell that attaches itself to the bag issupplied with a gentle flow of gaseous oxygen through the gas permeablemembrane of the bag, and it can discharge gaseous carbon dioxide orother waste gases through the bag. Meanwhile, the surrounding liquidprovides nutrients to the cell and carries off its waste products.

Many such gas permeable bags may be stacked to together and their inletsconnected by o-rings to form a convenient manifold. The stack can besubmerged in an aquarium-like enclosure that holds the bags tightly inthe stack. The o-rings, as well as beads or other spacers, serve to keepthe bags separated so there is a great amount of surface area for cellgrowth. There is a drop off in oxygen the farther one is away from abags, but the distance between the bags may be optimized so that acenter point between two bags has sufficient oxygen for cell growth.

Some embodiments of the present invention are related to a cell-growingsubstrate apparatus. The apparatus includes a pair of gas permeable,polymer sheets, an array of connection points between faces of thepolymer sheets, the connection points having a center-to-center spacingless-than-or-equal-to 2000 μm, a hermetically sealed edge that bonds aperimeter of the sheets together, enclosing the connection points in aninterior cavity between the sheets to form an expansion resistant bag,an inlet to the bag fluidly connected with the interior cavity, and abiocompatible coating over an outside of at least one of the gaspermeable sheets, the biocompatible coating having a thickness between0.01 μm and 1000 μm.

The center-to-center spacing of the connection points can be between 100μm and 1000 μm. The apparatus can further include an array of spacersconnecting the sheets at the connection points, the spacers having aheight sufficient to make the expansion resistant bag collapseresistant. A height of the spacers can be equal-to-or-greater-than onefifth the center-to-center spacing of the connection points. The spacerscan be integrally formed with one of the gas permeable sheets. Theapparatus can include a plurality of outside spacers abutting an outsideof the expansion resistant bag. The outside spacers can be porous tubesor spheres coated with a biocompatible coating. The outside spacers canbe integrally formed with one of the gas permeable sheets.

The biocompatible coating can include a parylene coating selected fromthe group including parylene N, parylene C, parylene D, and paryleneAF-4. The thickness of the parylene coating can be between 2 μm and 10μm. The thickness of the parylene coating can preferably be between 5 μmand 6 μm. A surface treatment area on the biocompatible coating can beconfigured to improve cell adhesion. The surface treatment area can be aproduct of an oxygen plasma-treatment, an ammonia plasma treatment, orboth an oxygen plasma-treatment and an ammonia plasma treatment. Acoating of agarose, collagen, lactic acid, laminin, poly-d-lysine, orpoly-l-lysine can be on the surface treatment area to further enhancecell growth.

The material of the gas permeable, polymer sheets can be a polymerselected from the group including of polydimethylsiloxane (PDMS),polyethylene, and polyurethane. The gas permeable, polymer sheet can beless than 200 μm thick. An outlet from the bag can be fluidly connectedwith the interior cavity. A tube having a lumen fluidly connected withthe inlet to or an outlet from the bag can be attached to the bag. Theinlet can pass through a hole in the hermetically sealed edge of thebag. The connection points in the array can be geometrically regularlyspaced apart, or irregularly spaced apart, from each other.

Some embodiments are related to a cell-growing substrate apparatus,including a sheet of polymeric open-cell foam having gas permeable skinsheets on two opposing sides, a hermetically sealed edge that bonds aperimeter of the gas permeable skin sheets together, enclosing theopen-cell foam in an interior cavity between the gas permeable skinsheets to form an expansion- and collapse-resistant bag, an inlet to thebag fluidly connected with the interior cavity, and a biocompatiblecoating over an outside of at least one of the gas permeable skinsheets, the biocompatible coating having a thickness of 0.01 μm to 1000μm. The sheet of polymeric open-cell foam can be 0.1 mm to 1.5 mm thick.

Some embodiments are related to a cell-growing substrate apparatus,including a watertight vessel, a stack of biocompatible material-coated,flat, expansion resistant gas permeable bags within the vessel, each baghaving an associated inlet to an interior cavity of the bag, and spacersbetween the expansion resistant gas permeable bags.

The gas permeable bags can be stacked in vertical planes. The stack caninclude at least one gas permeable bag folded over onto itself. Thestack can include at least one gas permeable bag coiled around itself.The apparatus can further include o-rings that space each bag apart andsealing the inlets of the bags to each other.

Some embodiments are related to a method of manufacturing a cell growingapparatus. The method includes casting a pillared gas permeable sheethaving spacers on one side, the gas permeable sheet having a thicknesssufficient to allow gaseous molecular oxygen to permeate therethroughyet prevent liquid water from permeating therethrough at standard roomtemperature and atmospheric pressure, fabricating a second gas permeablesheet, joining the second gas permeable sheet to the pillared gaspermeable sheet with the spacers therebetween, hermetically sealing aperimeter of the gas permeable sheets together to form an expansionresistant bag, depositing a biocompatible coating on at least one sideof the expansion resistant bag, the biocompatible coating having athickness between 0.1 μm and 1000 μm, and treating the biocompatiblecoating in order to improve cell adhesion.

The pillared gas permeable sheet and second gas permeable sheet can becomprised of a polymer, and each sheet can have a thickness less than200 μm. The method can further include forming an inlet in the expansionresistant bag and/or applying adhesive to the second gas permeablesheet.

Some embodiments are related to a method of manufacturing a cell growingapparatus. The method includes intimately contacting a pair of gaspermeable, polymer sheets together, placing a heat-insulative maskhaving an array of through holes against the polymer sheets, the throughholes having a center-to-center spacing less-than-or-equal-to 2000 μm,pressing a heated iron against the heat-insulative mask opposite thepolymer sheets, a temperature of the heated iron and duration of thepressing sufficient to form an array of welds between the polymer sheetswhere exposed by the mask through holes, hermetically sealing aperimeter of the polymer sheets together to form a bag with an interiorportion, depositing a biocompatible coating on at least one side of thebag, the biocompatible coating having a thickness between 0.1 μm and1000 μm, and treating the biocompatible coating in order to improve celladhesion.

The through holes can be photolithographically formed in the mask suchthat they are tiny. The polymer sheets can include polydimethylsiloxane(PDMS), and the heat-insulative mask includes silicone rubber.

Some embodiments are related to a method of growing and harvestingcells. The method includes providing a stack of expansion resistant, gaspermeable, polymer bags, each bag having an inlet connected with amanifold, each bag having a biocompatible coating, the bags being spacedfrom one another by spherical beads, seeding cells into interstitialspaces between the beads and bags, flowing a liquid culture of nutrientsbetween the interstitial spaces, pressurizing molecular oxygen gas intothe expansion resistant bags through the manifold, waiting for the cellsto grow, the nutrients and molecular oxygen gas supporting growth of thecells, applying trypsin to the cells to cleave their hold on the bag orbeads, and flushing the cells from the beads.

The method can further include removing the bags from the beads. Themethod can further include infecting the cells with a virus, where thegrowth of the cells serves to replicate viruses, and separating thereplicated viruses from the flushed cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an isometric view of a gas permeable, expansion resistant,collapse resistant bag in accordance with an embodiment.

FIG. 1B illustrates cross section B-B of FIG. 1A.

FIG. 1C illustrates cross section C-C of FIG. 1A.

FIG. 2A is an isometric view of a gas-permeable bag with spacer tubes inaccordance with an embodiment.

FIG. 2B is an exploded view of the gas-permeable bag of FIG. 2A.

FIG. 3A is an isometric view of a gas permeable microfabricatedarray-welded bag in accordance with an embodiment.

FIG. 3B illustrates cross section B-B of FIG. 3A.

FIG. 4 is an isometric, exploded view of an open-cell foam gas permeablebag in accordance with an embodiment.

FIG. 5 illustrates multiple bags separated by spacer tubes in accordancewith an embodiment.

FIG. 6 illustrates multiple bags separated by beads in accordance withan embodiment.

FIG. 7 is an isometric view of a bioreactor vessel having stacked gaspermeable bags in accordance with an embodiment.

FIG. 8 is an isometric view of a bioreactor with interdigitated nutrientand gas delivery bags in accordance with an embodiment.

FIG. 9A is a cross section and illustrates a portion of amicrofabricated bag manufacturing process in accordance with anembodiment.

FIG. 9B illustrates a portion of the manufacturing process of FIG. 9A.

FIG. 9C illustrates a portion of the manufacturing process of FIG. 9A.

FIG. 9D illustrates a portion of the manufacturing process of FIG. 9A.

FIG. 9E illustrates a portion of the manufacturing process of FIG. 9A.

FIG. 9F illustrates a portion of the manufacturing process of FIG. 9A.

FIG. 10A is a photograph of a prototype expansion resistant, collapseresistant bag in accordance with an embodiment.

FIG. 10B is a photograph of a corner of the prototype expansionresistant, collapse resistant bag of FIG. 10A.

FIG. 11 is a flowchart of a process in accordance with an embodiment.

FIG. 12 is a flowchart of a process in accordance with an embodiment.

FIG. 13 is a flowchart of a process in accordance with an embodiment.

DETAILED DESCRIPTION

A culture system (e.g. box, container, other parts) capable of very highdensity cell culture is described. The inventors recognized thatmolecular oxygen (O₂) is the most limiting metabolite for cell growthdue to its low solubility in cell media. By separating gas convectivedelivery from other nutrient delivery, it is possible to significantlyincrease oxygen delivery to cells without increasing detrimental shearforces.

Gas perfusable, gas permeable membrane sheets, formed into bags, providea high surface area while delivering sufficient oxygen and gas exchangefor high-density cell growth. These bags can be folded or stacked toachieve high surface area to volume ratios. Cells can be grown directlyon the surface of the membranes or on substrates sandwiched between themembranes.

While the use of separate membranes for oxygen delivery is useful, thereare other aspects including delivery and removal of other nutrients,solutions, cells, and viruses. For example, a gap between stackedmembranes can be perfused with solutions to deliver or remove componentsinto or out of the membrane stacks. The gap and rate of perfusion can bechosen so as to maintain suitable shear rates within the device. Inanother example, a network of tubes or channels can be employedthroughout the system for delivery separate from the gas supply. Thetubes or channels may contain pores to allow various sized particlesranging from molecules to cells to pass into or out of the tubing. Thisis a means by which cells can be seeded within the device and/or viruscan be delivered to infect cells. In another example, pores can beformed in the membranes themselves to provide a means of perfusing thestacked membranes normal to their surfaces. In this arrangement the flowinduces minimal shear forces on cells because the direction of flow isnot parallel to the cells; nutrients then reach the cells throughdiffusion away from these pores.

When seeding cells, both sides of the membranes can be equally seeded.This can be achieved by orienting the membranes and flow parallel to thegravitational field. Since cells naturally settle in media, a perfusionof media equal in magnitude but opposite in direction to the settlingvelocity can be applied so that the net velocity of the cells withrespect to the membranes is close to zero. By symmetry, the cells areequally likely to adhere to either side of the membranes resulting inuniform seeding.

In terms of membrane materials, those with high gas transmissionproperties are most suitable. This can be accomplished by usingmaterials with high gas permeation (a function of solubility anddiffusion rate) such as silicones, including polydimethylsiloxane(PDMS).

Alternatively, it can be accomplished by using very thin membranesbecause gas transmission rates are generally inversely proportional tomembrane thickness. At very thin dimensions, the permeability of thematerial may also increase significantly, such as is observed with thinparylene (<10 μm). Using this fact, polymers that are conventionallythought of as barriers to gases can become suitably transmissive. Porousmembrane materials may also be used since the blow point of small porescan be sufficiently high to allow pressure driven from with the hollowmembrane without causing bubbling of gas through the surface. Acombination of approaches can also be used to accomplish the desiredhigh gas transmission properties of the membranes.

Surface treatment of the bags can achieve cell adhesion andproliferation. A broadly applicable approach is to coat the membranewith a thin layer of parylene and plasma etch it, using an oxygen plasmatreatment and/or an ammonia plasma treatment, to make it hydrophilic.

Additional methods include coating with proteins (e.g. agarose,collagen, fibronectin, fibrin) or other coatings (e.g., lactic acid,laminin, poly-D-lysine, or poly-L-lysine).

TERMS

A “gas permeable” or “semipermeable” material includes that which allowsa gas to permeate through but prevent liquid water from permeating, oras otherwise applicable and known in the art. Permeability can bemeasured at standard room temperature (i.e., 25° C.) and atmosphericpressure or other applicable temperatures and pressures. For example astandard temperature for cultures of mammalian cells is 37° C. Forthermophiles, temperatures can go up to 122° C. Pressures can beelevated from or less than standard atmospheric pressure, such as withhydrostatic pressure from being submerged. A gas may be molecularoxygen, carbon dioxide, carbon monoxide, common air, or as otherwiseapplicable.

A “pillar” or “micropillar” includes a column or other structure thatextends perpendicularly from a surface, or as otherwise known in theart. A “micropillar” includes a pillar that is small and is not limitedto pillars that are on the scale of micrometers (microns) ormicro-inches.

An “array of spacers” includes a geometrically regular or irregularpattern of micropillars or other spacers with a height andcenter-to-center spacing of the spacers configured to keep the sheets ata fixed distance from one another, or as otherwise known in the art.

A “collapse resistant bag” includes a bag that has spacers inside itsuch that internal surfaces of opposite sides are prevented fromtouching each other in normal operation.

An “expansion resistant bag” includes a bag that has internal weldpoints, connected spacers, or other connections that prevent the bagfrom becoming shaped like a balloon when pressurized, or as otherwiseknown in the art. An expansion resistant bag may lay substantially flat.It may have convex and concave recesses and curves.

“Hermetically sealed” simply means sealed to be airtight, or at leastnot permeable to liquid but perhaps permeable to gas, or as otherwiseknown in the art.

Open cell foam that may be compatible with certain embodiments includesAtlantic Gasket Corp. Style AG1300-S or AG1300-SM open cell siliconefoam, with skin in accordance with ASTM D3183, and McMaster-Carrsilicone foam sheets.

FIGS. 1A-1B illustrate a gas permeable, expansion resistant, collapseresistant bag in accordance with an embodiment. System 100 includes apair of gas permeable, PDMS polymer sheets 102 and 103. The sheets'perimeters have been hermitically sealed around their edges to formexpansion resistant bag 101 with interior cavity 113.

Bottom sheet 103 has integrally formed edge 112 around its perimeter andspacers, which are microfabricated pillars 104, along one face.Micropillars 104 are in a geometrically regular array of columns androws. Each column of micropillars is separated by distance 110, and eachrow of micropillars is separated by distance 111. In some embodiments,the distances are different; in others, the distances are the same.

Other configurations are envisioned, including geometrically regulararrays with staggered rows or columns, circular arcs of pillars, subshapes, and other patterns, as well as geometrically irregular arrays,such as randomly distributed speckles of pillars.

During fabrication, sheet 102 or the tops of the micropillars and edgeof sheet 103 were coated with uncured polymer, and then sheet 102 isplaced atop sheet 103 before curing. The sheets 102 and 103 and middleportion 112 form a collapse resistant sandwich of layers.

On the outside of sheets 102 and 103 is parylene biocompatible coating115. The biocompatible coating has a thickness between 0.01 μm and 1000μm, which may control the gas permeability of the underlying sheet. Thatis, the PDMS may be permeable to liquids at its nominal thickness;however, the thin coating of parylene prevents liquids from goingthrough them but allowing gas, thus rendering the sheets gas permeable.

Surface treatment 116 covers a portion of biocompatible coating 115. Theparylene was subject to an oxygen plasma treatment and ammonia plasmatreatment in order to improve cell adhesion.

In FIG. 1C, going from top to bottom, top biocompatible coating 115 hasthickness 105, sheet 102 has thickness 106, micropillars 104 have equalheights 107, sheet 103 has thickness 108, and bottom biocompatiblecoating 115 has thickness 109. The thickness of the biocompatiblecoating, if it is parylene N, parylene C, parylene D, or parylene AF-4,is between 2 μm and 10 μm, preferably between 5 μm and 6 μm.

Height 107 of micropillar spacers 104 is more than one fifth thecenter-to-center spacing of the micropillar connection points. Thisheight prevents opposing sheets 102 and 103 from bending inward andclinging to each other within the internal cavity, helping to make thebag collapse resistant.

Inlet 114 is formed as a hole through top sheet 102 and biocompatiblelayer 115. Inlet 114 may be used for supplying gas, or removing gas,from bag 101.

FIGS. 2A-2B illustrate a gas-permeable bag with spacer tubes inaccordance with an embodiment. Bag 201 includes biocompatible layers 215over sheets 202 and 203 with middle layer 212. Inlet 214 is formed insheet 202 and its biocompatible layer 215, with surface treatment 216shown on biocompatible layer 215. Also shown is a cutaway view to insidethe bag where micropillars 204 keep the bag from collapsing.

Below bag 201 are external spacer tubes 217. Each spacer tube 217 hasmultiple holes 218 that allow fluid, nutrients, seed cells, and othermaterial to flow out of them. The spacer tubes resemble a French drainor garden soaker hose. However, the spacer tubes are microfabricated,allowing cell-sized or smaller materials to be distributed relativelyevenly. Thus, if bag 201 were stacked with other bags and separated byspacer tubes, the spacer tubes could be used to evenly seed cells,spread nutrients, and accumulate discharged waste.

FIGS. 3A-3B illustrate a gas permeable microfabricated array-welded bag.In system 300, bag 301 includes sheets and no additively formed innerpillars to keep apart the sheets. Instead, the sheets are kept apart bymild gas pressure inside of the bag.

An array of connection points 319 was created by pressing a heated ironagainst the sheets with a heat-insulative mask therebetween, such thatthe only areas that are welded are those exposed by through holes in themask. The areas around the welds allow air to move freely. This slightlyresembles an inflatable swimming pool air mattress. However, theconnection points of the embodiment are less-than-or-equal-to (≤) 2000μm apart from one another. In some embodiments, the center-to-centerspacing of the connection point welds are between 100 μm and 1000 μm.Such tiny spacings can be formed using microfabrication techniques, suchas photolithography. Further, the polymer sheets are less than 200 μmthick in the exemplary embodiment.

Perimeter edge 312 is hermetically sealed to form bag 301, and hole 314is formed in at least one of the sheets; here, hole 314 is formed insheet 302. Biocompatible layers 315 are on the outside of sheets 302 and303.

When provided oxygen through inlet hole 314, interior portion 313 opensup and expands. If the pressure of provided air is great enough, even asubmerged bag's dry interior portions 313 open up so that the gas canflow freely to the farthest reaches of the bag. The oxygen then evenlypermeates through sheets 302 and 303, through biocompatible layers 315,to consuming cells outside.

FIG. 4 illustrates an open-cell foam gas permeable bag. In system 400, a0.1 mm to 1.5 mm thick mat of polymeric open-cell foam 421 has gaspermeable skin sheets 420 on opposing sides. That is, naturally formedskins on opposing sides of the foam are permeable to gas. The foam'sperimeter edge 412 (shown exploded away from the central foam area inthe figure) is hermitically sealed so as to bond skin sheets 420together and enclose the open-cell foam in an interior cavity. The foamprevents the bag from expansion and collapse. Thus, anexpansion-resistant, collapse-resistant bag is formed.

Biocompatible coating 415 is layered over the outside skins 420 of thebag, and it has a thickness of 0.01 μm to 1000 μm.

FIG. 5 illustrates multiple bags separated by spacer tubes. Each bag 501is shown in cross section, and each bag has hole 514 through bothsheets. O-rings 525, shown in cross-section, space apart the bags at apredetermined distance and seal the insides of the bags to one another.Air or oxygen may be supplied through the o-rings so that the gas flowsinto each of the bags.

Besides the o-rings, also spacing apart bags 501 are tube spacers 517,which are shown in cross section. Tube spacers 517 may have holes,slits, or other portions through which nutrients, seed cells, or othermaterials may be sent to be evenly distributed.

In some embodiments, tube spacers 517 may be oriented so that theyextend up and down as opposed to how they are shown (i.e., in and out ofthe page). In other embodiments, tube spacers may snake around betweenthe bags.

FIG. 6 illustrates multiple bags separated by beads. Each bag 601 isshown in cross section, and each bag has hole 614 through both sheets.O-rings 625, shown in cross-section, space apart the bags at apredetermined distance and seal the insides of the bags to one another

Bags 601 are kept separated by spherical beads 626. The beads providemore surface area for cell growth than just the bags. Cells may beseeded within interstitial spaces 627 between the beads and the bags,and a liquid culture of nutrients can be flowed therethrough. The beads'outsides may be treated for better cell adhesion, to hold nutrients,etc. Meanwhile, gas is provided into the dry interiors of the bagsthrough the o-ring manifold.

After the cells have grown, the bags may be removed from the beads bypulling the bags out or draining the beads from in between the bags. Thebeads may then be processed to remove the cells from them. For example,trypsin may be added to cleave the bonds between the cells and the beads(and bags), or the beads may be vigorously washed so that the cells comeoff.

FIG. 7 is an isometric view of a bioreactor vessel having stacked gaspermeable bags. In system 700, bags 701 are spaced apart by apredetermined amount, and they are hung in vertical planes so that theirwalls are vertical with respect to gravity. In other configurations, along bag may be coiled around itself, like a paper towel roll.

Watertight vessel 730 contains the bags and immerses them in a liquidculture. The liquid may be circulated gently while a manifold deliversoxygen or air to the bags. Openings to the bags may be connected to oneanother, such as in FIGS. 5-6, or tubes with lumens connecting to theinsides of the bags may be connected with a common manifold.

FIG. 8 is an isometric view of a bioreactor with interdigitated nutrientand gas delivery bags. In system 800, structure 831 of gas bag membranes813 is interleaved with nutrient delivery membranes 826. This forms anextremely compact structure where gas and nutrients are providedthroughout the bioreactor.

The bioreactor may be contained in a vessel (not shown in the figure) tocontain liquid and cell nutrients.

FIGS. 9A-9F illustrate a microfabricated bag manufacturing process. InFIG. 9A, a continuous dry film mold with elevated edges is set atop aflat silicon substrate. In FIG. 9B, the dry film mold isphotolithographically etched to create through holes in the dry film. InFIG. 9C, PDMS is applied over the mold and allowed to cure. The resultis a PDMS sheet with micropillars and a tall edge around its perimeter.

In FIG. 9D, PDMS is applied over a mold similar to that in FIG. 9A andallowed to cure. The result is a flat sheet of PDMS. The two sheet ofPDMS are then brought together, pillar side of the first sheet betweenthem, and glued using a thin PDMS layer that then cures to create a bag.The micropillars prevent expansion and prevent collapse of themicrofabricated bag.

In FIG. 9E, a tube is inserted in a side of the bag so that a lumen ofthe tube connects through a hole in the hermetically sealed edge of thebag with an interior cavity of the bag. In FIG. 9F, parylene is coated,using chemical vapor deposition (CVD), over top, bottom, and side of thebag. The parylene presents a clean, biocompatible surface for cells. Theparylene also controls the permeability of the sheets. The parylene canbe plasma treated for better cell adhesion properties.

FIGS. 10A-10B are microscope photographs of a prototype expansionresistant, collapse resistant bag. The bag is clear so that one may seethrough to its micropillars within its interior. Evident in the figuresis a geometrically regular pattern, columns and rows, of micropillarswithin the bag. The rows and columns are around 100 μm apart Also shownare the bag's sealed edges, which show up dark in the micrograph.

The hollow PDMS bags/membranes (120 μm thick membranes with a 120 μmcentral gap) are coated with a thin layer (˜0.5 μm) of plasma treatedparylene C for cell adhesion. The edges of the bags are plumbed withtubing and connected to a regulated oxygen supply to perfuse the hollowportion of the bag with oxygen. Oxygen within this gap is able todiffuse through the PDMS membrane out into the surrounding media tonourish cells. Cells can be grown directly on the membranes with ratescomparable to tissue culture flasks.

FIG. 11 is a flowchart of process 1100 in accordance with an embodiment.In operation 1101, a pillared gas permeable sheet is cast with spacerson one side. The gas permeable sheet has a thickness sufficient to allowgaseous molecular oxygen to permeate therethrough yet prevent liquidwater from permeating therethrough at standard room temperature andatmospheric pressure. In operation 1102, a second gas permeable sheet isfabricated. In operation 1103, an adhesive is applied to the second gaspermeable sheet. In operation 1104, the second gas permeable sheet isjoined to the pillared gas permeable sheet with the spacerstherebetween. In operation 1105, a perimeter of the pillared and secondgas permeable sheets is hermetically sealed together to form acollapse-resistant bag. In operation 1106, an inlet in thecollapse-resistant bag is formed. In operation 1107, a biocompatiblecoating is deposited on at least one side of the bag, the biocompatiblecoating having a thickness between 0.1 μm and 1000 μm. In operation1108, the biocompatible coating is treated in order to improve celladhesion.

FIG. 12 is a flowchart of process 1200 in accordance with an embodiment.In operation 1201, a pair of gas permeable, polymer sheets areintimately contacted together. In operation 1202, a heat-insulative maskhaving an array of through holes is placed against the polymer sheets(on one side), the through holes having a center-to-center spacing ofless-than-or-equal-to 1000 μm. In operation 1203, a heated iron ispressed against the heat-insulative mask opposite the polymer sheets, atemperature of the heated iron and duration of the pressing sufficientto weld portions of the polymer sheets together that are exposed by themask through holes. In operation 1204, a perimeter of the polymer sheetsare hermitically sealed together to form a bag. In operation 1205, abiocompatible coating is deposited on at least one side of the bag, thebiocompatible coating having a thickness between 0.1 μm and 1000 μm. Inoperation 1206, the biocompatible coating is treated in order to improvecell adhesion.

FIG. 13 is a flowchart of process 1300 in accordance with an embodiment.In operation 1301, a stack of expansion resistant, gas permeable polymerbags is provided, each bag having an inlet connected with a manifold,each bag having a biocompatible coating, the bags being spaced from oneanother by spherical beads. In operation 1302, cells are seeded intointerstitial spaced between the beads and bags. In operation 1303, aliquid culture of nutrients is flowed between the interstitial spaces.In operation 1304, molecular oxygen gas is pressurized into theexpansion resistant bags through the manifold. In operation 1305, onewaits for the cells to grow and multiply, the nutrients and molecularoxygen gas supporting growth of the cells. In operation 1306, trypsin isapplied to the cells. In operation 1307, the cells are flushed from thebeads.

The invention has been described with reference to various specific andillustrative embodiments. However, it should be understood that manyvariations and modifications may be made while remaining within thespirit and scope of the following claims.

What is claimed is:
 1. A compact cell-growing apparatus comprising: awatertight vessel; a stack of biocompatible material-coated, flat,expansion resistant gas permeable bags within the vessel, each baghaving an associated inlet to an interior cavity of the bag; and spacersbetween the expansion resistant gas permeable bags, wherein each bagcomprises: a pair of gas permeable, polymer sheets; an array ofconnection points between faces of the polymer sheets; a hermeticallysealed edge that bonds a perimeter of the sheets together, enclosing theconnection points in an interior cavity between the sheets to form anexpansion resistant bag; and an inlet to the bag fluidly connected withthe interior cavity.
 2. The apparatus of claim 1 wherein the gaspermeable bags are stacked in vertical planes.
 3. The apparatus of claim1 wherein the stack includes at least one gas permeable bag folded overonto itself.
 4. The apparatus of claim 1 wherein the stack includes atleast one gas permeable bag coiled around itself.
 5. The apparatus ofclaim 1 further comprising: o-rings spacing each bag apart and sealingthe inlets of the bags to each other.
 6. The apparatus of claim 1wherein each bag comprises: the connection points have acenter-to-center spacing less-than-or-equal-to 2000 μm.
 7. The apparatusof claim 1 wherein each bag further comprises: a biocompatible coatingover at least one side of the gas permeable sheets, the biocompatiblecoating having a thickness between 0.01 μm and 1000 μm.
 8. The apparatusof claim 6 wherein the center-to-center spacing of the connection pointsis between 100 μm and 1000 μm.
 9. The apparatus of claim 1 wherein eachbag further comprises: an array of spacers connecting the sheets at theconnection points, the spacers having a height sufficient to make theexpansion resistant bag collapse resistant.
 10. The apparatus of claim 9wherein a height of the spacers is equal-to-or-greater-than one fifththe center-to-center spacing of the connection points.